Ceramide Synthesis Is Modulated by the Sphingosine AnalogFTY720 via a Mixture of Uncompetitive and NoncompetitiveInhibition in an Acyl-CoA Chain Length-dependent Manner*

Received for publication, September 25, 2008, and in revised form, March 30, 2009 Published, JBC Papers in Press, April 8, 2009, DOI 10.1074/jbc.M807438200

Sujoy Lahiri‡, Hyejung Park§, Elad L. Laviad‡, Xuequan Lu¶, Robert Bittman¶, and Anthony H. Futerman‡1

From the ‡Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel, the §School of Biology andPetit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332-0230, and the¶Department of Chemistry and Biochemistry, Queens College of the City University of New York, Flushing, New York 11367-1597

FTY720, a sphingosine analog, is in clinical trials as an immu-nomodulator. The biological effects of FTY720 are believed tooccur after its metabolism to FTY720 phosphate. However, verylittle is known about whether FTY720 can interact with andmodulate the activity of other enzymes of sphingolipid metabo-lism. We examined the ability of FTY720 to modulate de novoceramide synthesis. In mammals, ceramide is synthesized by afamily of six ceramide synthases, each of which utilizes arestricted subset of acyl-CoAs. We show that FTY720 inhibitsceramide synthase activity in vitro by noncompetitive inhibitiontoward acyl-CoA and uncompetitive inhibition toward sphinga-nine; surprisingly, the efficacy of inhibition depends on the acyl-CoA chain length. In cultured cells, FTY720 has amore complexeffect, with ceramide synthesis inhibited at high (500 nM to 5�M)butnot low (<200nM) sphinganine concentrations, consist-ent with FTY720 acting as an uncompetitive inhibitor towardsphinganine. Finally, electrospray ionization-tandem massspectrometry demonstrated, unexpectedly, elevated levels ofceramide, sphingomyelin, and hexosylceramides after incuba-tion with FTY720. Our data suggest a novel mechanism bywhich FTY720 might mediate some of its biological effects,which may be of mechanistic significance for understanding itsmode of action.

FTY720 (2-amino-(2-2-[4-octylphenyl]ethyl)propane 1,3-diolhydrochloride), also known as Fingolimod, is an immunosup-pressant drug currently being tested in clinical trials for organtransplantation and autoimmune diseases such as multiplesclerosis (1). FTY720 is a structural analog of sphingosine, a keybiosynthetic intermediate in sphingolipid (SL)2 metabolism(see Fig. 1). In vivo, FTY720 is rapidly phosphorylated by sphin-gosine kinase 2 (2, 3) to form FTY720 phosphate (FTY720-P),an analog of sphingosine 1-phosphate (S1P) (see Fig. 1A).

FTY720-P binds to S1P receptors (S1PRs) (4, 5) and therebyinduces a variety of phenomena such as T-lymphocyte migra-tion from lymphoid organs (6–9); accordingly, FTY720 treat-ment results in lymphopenia as lymphocytes (especiallyT-cells) become sequestered inside lymphoid organs (10–12).The ability of FTY720 to sequester lymphocytes has stimulatedits use in treatment of allograft rejection and autoimmune dis-eases (13), and FTY720 is currently under phase III clinical tri-als for treatment of relapsing-remitting multiple sclerosis (14).Apart from the binding of FTY720-P to S1PRs, the ability of

FTY720 to inhibit S1P lyase (15) (see Fig. 1B), and its inhibitoryeffect on cytosolic phospholipase A2 (16), whose activity can bemodulated by ceramide 1-phosphate (17), little is known aboutwhether FTY720 or FTY720-P can modulate the activity ofother enzymes of SL metabolism. Because FTY720 is an analogof sphingosine, one of the two substrates of ceramide synthase(CerS) (see Fig. 1), we now examine whether FTY720 canmod-ulate CerS activity. CerS utilizes fatty acyl-CoAs to N-acylatesphingoid long chain bases. Six CerS exist in mammals, each ofwhich uses a restricted subset of acyl-CoAs (18–23). We dem-onstrate that FTY720 inhibits CerS activity and that the extentof inhibition varies according to the acyl chain length of theacyl-CoA substrate. Surprisingly, FTY720 inhibits CerS activitytoward acyl-CoA via noncompetitive inhibition and towardsphinganine via uncompetitive inhibition. Finally, the mode ofinteraction of FTY720 with CerS in cultured cells depends onthe amount of available sphinganine. Together, we show thatFTY720 modulates ceramide synthesis, which may be of rele-vance for understanding its biological effects in vivo and its rolein immunomodulation.

EXPERIMENTAL PROCEDURES

Materials—D-Erythro-[4,5-3H]Sphinganine (80 Ci/mmol),FTY720, (R)-FTY720-P and (R)-FTY-phosphonate were syn-thesized as described (24, 25). Fatty acyl-CoAs, ceramides, di-methylsphingosine, lysophosphatidic acid, and the internalstandards for liquid chromatography electrospray ionization-tandem mass spectrometry (ESI MS/MS) were from AvantiPolar Lipids (Alabaster, AL). Sphinganine was from Matreya(Pleasant Gap, PA). Silica gel 60 TLC plates were from Merck.Defatted bovine serumalbumin (BSA), suramin, S1P, and a pro-tease inhibitor mixture were from Sigma-Aldrich. Dulbecco’smodified Eagle’s medium was from Invitrogen or Sigma-Al-

* This work was supported, in whole or in part, by National Institutes of HealthGrant GM076217 (to A. M. and A. H. F.), and HL-083187 (to R. B.). This workwas also supported by Israel Science Foundation Grant 1404/07 and byfunds from the Minerva Foundation (to A. H. F.).

1 Joseph Meyerhoff Professor of Biochemistry at the Weizmann Institute ofScience. To whom correspondence should be addressed. Tel.: 972-8-9342704; Fax: 972-8-9344112; E-mail: tony.futerman@weizmann.ac.il.

2 The abbreviations used are: SL, sphingolipid; BSA, bovine serum albumin;CerS, ceramide synthase; ESI-MS/MS, electrospray ionization-tandem massspectrometry; FTY720-P, FTY720 phosphate; HEK, human embryonic kid-ney; HEPG2, hepatocellular liver carcinoma; RBL, rat basophilic leukemia;S1P, sphingosine 1-phosphate; S1PR, S1P receptor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 24, pp. 16090 –16098, June 12, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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drich. All of the solvents were of analytical grade and were pur-chased from Biolab (Jerusalem, Israel).Cell Culture and Transfection—Human embryonic kidney

(HEK) 293T cells, RBL-2H3mast cells, and hepatocellular livercarcinoma (HEPG2) cells were cultured inDulbecco’smodifiedEagle’s medium supplemented with 10% fetal calf serum, 100IU/ml penicillin, and 100 �g/ml streptomycin. HEK cells weretransfected with human CerS genes (26) using the calciumphosphate method (0.25 �g of plasmid/cm2 of culture dish),which gave �90% transfection efficiency.CerS Assay—The cells were removed fromculture dishes using

trypsin (0.25%, w/v) followed by centrifugation (4min, 150� gav).The cell pellets were washed with phosphate-buffered saline andhomogenized in20mMHEPES-KOH,pH7.4, 25mMKCl, 250mM

sucrose, and 2 mM MgCl2 containinga protease inhibitor mixture. Proteinwas estimated using the Bradfordprotein assay (Bio-Rad). CerS activitywas assayed essentially as described(26). Briefly, cell homogenates wereincubated with [4,5-3H]sphinganine/sphinganine/20�Mdefatted BSA andfatty acyl-CoA for 20 min at 37 °C.Stearoyl-CoA (C18-CoA) was usedfor CerS1 and 4 (18, 19), lignoceroyl-CoA (C24-CoA) or behenoyl-CoA(C22-CoA) was used for CerS2 (22),and palmitoyl-CoA (C16-CoA) wasused for CerS5 (19). The amount ofhomogenate used for each CerS wastaken so that the reaction ratewas lin-earwith respect to the amountof pro-tein (26). The reactions were termi-nated using chloroform/methanol(1/2; v/v), and the lipids wereextracted (27). Lipids were separatedby TLC using chloroform/metha-nol/2 M ammonium hydroxide (40/10/1; v/v/v) as developing solvent.[3H]Lipids were identified usingauthentic standards, visualized usinga phosphorimaging screen (Fuji,Tokyo, Japan), and recovered fromthe TLC plates by scraping the silicadirectly into scintillation vials con-taining 1 ml of methanol and 5 ml ofOptimaGold scintillation fluid (Pack-ard, Downers Groove, IL). Radioac-tivity was determined using aPackard 2100 beta radiospectrometerequipped with the TransformedSpectral Index of the External Stand-ard/Automatic Efficiency Controlprogram for quench correction.Metabolic Labeling—HEK and

RBL-2H3 cells were incubated with25 �M FTY720 for 1.5 h followed byincubation with [4,5-3H]sphinganine

or [4,5-3H]sphinganine/sphinganine during the last 30 min of theincubation period; when transfected HEK cells were used, theywere incubatedwith FTY720 and sphinganine 24 h after transfec-tion. The cells were removed from the culture dishes by scrapinginto methanol, and the lipids were extracted (28). The lipids wereseparated by TLC using chloroform/methanol (50:3.5; v/v) asdeveloping solvent, identified, and quantified as above.ESI-MS/MS—SL analyses by ESI-MS/MS were conducted

using a PE-Sciex API 3000 triple quadrupole mass spectrom-eter and an ABI 4000 quadrupole linear ion trap mass spec-trometer as described (18, 19, 29, 30). HEK cells were har-vested by trypsinization, collected by centrifugation, washedtwice with ice-cold phosphate-buffered saline, and lyophi-lized. The samples were spiked with a SL internal standard

FIGURE 1. SL structure and metabolism. A, structures of SLs and SL analogs used in this study. B, metabolicinter-relationships between SLs and the metabolism of FTY720. The enzymes are denoted in italics. LPP3, lipidphosphate phosphatase 3; LPP1�, lipid phosphate phosphatase 1�.

Characterization of Ceramide Synthesis Inhibition by FTY720

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mixture (Avanti Polar Lipids), then extracted, and analyzedby liquid chromatography ESI-MS/MS.

RESULTS

Inhibition of CerS Activity—To determine whether FTY720inhibits CerS activity in vitro, homogenates from HEK cellsoverexpressing CerS2 were incubated with increasing con-centrations of FTY720. Significant inhibition of CerS2 activ-ity was observed (see Fig. 2A). In contrast, another sphingo-sine analog, dimethylsphingosine (Fig. 1), had no effect onCerS2 (Fig. 2A) or CerS4 and 5 activity (not shown). The lackof effect of dimethylsphingosine also acts as an importantcontrol, demonstrating that the inhibition of CerS2 byFTY720 does not occur via a nonspecific mechanism. Forinstance, it is conceivable that FTY720 could displace sphin-ganine from the complex it forms with BSA (which acts as alipid carrier in the reaction mixture; see Refs 24 and 26), thusrendering sphinganine inaccessible for the reaction. Basedon these results, we conclude that FTY720 specifically inhib-its CerS2.We have recently shown that CerS2 contains an S1PR-like

motif via which S1P inhibits CerS2 activity (22). To determinewhether FTY720-P, an analog of S1P (Fig. 1A), can also inhibitCerS2 via this motif, homogenates from CerS2-overexpressingcells were incubated with FTY720-P. No inhibition was

observed, whereas S1P significantly inhibited CerS2 (Fig. 2B),as reported previously (22). This result also demonstrates thatFTY720 itself (Fig. 2A) and not FTY720-P, is responsible forCerS2 inhibition. Similarly, FTY720-P had no effect on CerS5activity (data not shown). Likewise, lysophosphatidic acid didnot inhibit CerS2 (Fig. 2B), confirming the specificity of theCerS2 S1P receptor-like motif for S1P.We next examined the level of inhibition of FTY720 using

different acyl-CoAs for ceramide synthesis (Fig. 3). In threedifferent cell lines, namely HEK, RBL-2H3 mast cells, and hep-atocellular liver carcinoma (HEPG2) cells, the EC50 of inhibi-tion was lowest using C18-CoA, which gave an EC50 value ofbetween 35 and 47�M (Fig. 3). In contrast, the EC50 values usingeither C16-CoA or C24-CoA were considerably higher (Fig. 3).The dependence on acyl-CoA chain length was further exam-ined by comparing the EC50 value usingHEK cells overexpress-ing either CerS5 or CerS1, which use C16- and C18-CoA,respectively (18, 19). FTY720 inhibited CerS1 activity to agreater extent than CerS5 (Fig. 4A). Finally, we analyzed theactivity of CerS4 in HEK cells using C18- or C20-CoA; CerS4can utilize either of these acyl-CoAs for ceramide synthesis(19). There was a significant difference in the EC50, withvalues of 32 and 65 �M for C18- and C20-CoA, respectively(Fig. 4B). Together, these results suggest that the efficacy ofinhibition of CerS by FTY720 depends on the acyl-CoAchain length. This result is unexpected because FTY720 is asphingosine analog (Fig. 1A), and therefore we assumed thatthe chain length of the acyl-CoA would not be relevant forCerS inhibition by FTY720.We also examined whether FTY720 could be acylated by

CerS. HEK cells overexpressing CerS4 or CerS5were incubatedwith 1-[14C]stearoyl-CoA or 1-[14C]palmitoyl-CoA, respec-tively. Although [14C]stearoyl-sphinganine (C18-ceramide)and [14C]palmitoyl-sphinganine (C16-ceramide) were bothsynthesized and their formation significantly inhibited in thepresence of FTY720, no additional radioactive bands could bedetected by TLC after incubation with FTY720 (data notshown).Kinetic Analysis—We next examined the inhibition kinetics

of FTY720 in homogenates from HEK cells overexpressingCerS4. Because FTY720 is a structural analog of the sphingoidlong chain base (Fig. 1A), we assumed that FTY720would act asa competitive inhibitor toward sphinganine. Surprisingly, how-ever, FTY720 was an uncompetitive inhibitor toward sphinga-nine (Fig. 5A).In contrast, FTY720 acted as a noncompetitive inhibitor

toward C18-CoA (Fig. 5B). The inhibition of FTY720 towardC18-CoA was allosteric (Fig. 5B) under the normal reactionconditions used to assay CerS (i.e. using defatted BSA as lipidcarrier, see above). However, when the assay was performed inthe absence of defatted BSA, classical noncompetitive inhibi-tion was obtained using the Lineweaver-Burk plot (Fig. 5B,inset). These data indicate that FTY720 has a complex mode ofinhibition onCerS in vitro, acting as an uncompetitive inhibitortoward sphinganine and a noncompetitive inhibitor towardacyl-CoA.Modulation of Ceramide Synthesis in Cultured Cells—To

determine whether FTY720 also inhibits ceramide synthesis

FIGURE 2. Effect of FTY720 on CerS2 activity. CerS2 activity was assayed inhomogenates (100 �g of protein) from CerS2-overexpressing cells using 0.25�Ci of [4,5-3H]sphinganine, 15 �M sphinganine, 20 �M defatted BSA and 50�M C22-acyl-CoA, with FTY or dimethylsphingosine (A) or FTY720-P, lyso-phosphatidic acid, or S1P (B). The specific activity of CerS2 in the absence ofinhibitors was 84 � 4.6 pmol ceramide formed per min per mg of protein. Thedata in A is a typical experiment for HEK cells overexpressing CerS2, and sim-ilar results were obtained for cells overexpressing CerS4 and 5. In B, the resultsare the means � S.D. for FTY720-P and S1P (n � 4) and the means (n � 2) forlysophosphatidic acid.

Characterization of Ceramide Synthesis Inhibition by FTY720

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in cultured cells, mock-transfected HEK cells were incu-bated with 25 �M FTY720 and then metabolically labeledwith 1 �Ci of [4,5-3H]sphinganine/2 �M sphinganine. Signif-icant inhibition of ceramide synthesis was observed underthese conditions (Fig. 6A). Similarly, ceramide synthesis wasinhibited by FTY720 in CerS1- and CerS4-overexpressingHEK cells (Fig. 6A). FTY720 also inhibited ceramide synthe-sis in RBL-2H3 cells (Fig. 6A), demonstrating that the inhi-bition of ceramide synthesis by FTY720 also occurs in cells ofimmune origin.The amount of [4,5-3H]sphinganine/sphinganine used in

these metabolic labeling experiments (�12 nmol/mg protein)was significantly higher than endogenous sphinganine levels inHEK cells (�6 pmol/mg protein) (19). Because FTY720 has acomplex mode of inhibition of CerS activity (see above), weexamined the concentration dependence of the effect ofFTY720 versus sphinganine concentration. At high sphinga-nine concentrations, i.e. 500 nM to 5 �M, FTY720 (25 �M) sig-nificantly inhibited ceramide synthesis (Fig. 6B; cf. Fig. 6A).However, at lower sphinganine concentrations (�200 nM), noinhibition of ceramide synthesis was observed. Indeed,although not statistically significant, a tendency toward anincrease in ceramide synthesis was detected at the lowest (�1

nM) sphinganine concentrations.3 Thus, themodulation of cer-amide synthesis by FTY720 in cultured cells depends on theratio of the concentrations of sphinganine to FTY720.This unexpected result was supported by data obtained using

ESI-MS/MS to determine total ceramide, sphingomyelin, andhexosylceramide levels in HEK cells and the fatty acid compo-sition of these SLs (Fig. 7). Upon incubation of HEK cells withFTY720 for 90min (the same time of incubation as used for themetabolic labeling experiments) (Fig. 6A), an increase in cera-mide levels was observed, with no change in endogenous sph-inganine levels (Fig. 7); similar increases were observed forhexosylceramide and sphingomyelin. Moreover, levels of C18-C22-ceramide were significantly increased, as were levels ofC18-C22-hexosylceramide and C18-C22-sphingomyelin. Thisresult is consistent with a complex mode of interaction ofFTY720with CerS, perhaps involving an allosteric element thatis not preserved in vitro, whereby ceramide synthesis is stimu-lated in cells despite its inhibition by FTY720 in vitro.

3 At the lowest [4,5-3H]sphinganine concentrations, the amount of [3H]cera-mide formed (5,000 dpm) is extremely low, even though the specific activ-ity of [4,5-3H]sphinganine is high (80 Ci/mmol). Thus, these experimentsare difficult to perform to a high degree of experimental accuracy.

FIGURE 3. Inhibition of CerS activity by FTY720 using various acyl-CoAs. CerS activity was assayed in homogenates (100 �g of protein) from HEK, RBL-2H3,and HEPG2 cells using 0.25 �Ci of [4,5-3H]sphinganine, 15 �M sphinganine, 20 �M defatted BSA, and 50 �M of acyl-CoA, as indicated. The results are the means �S.D. (n � 3) for HEK cells and the means � S.D. of two independent experiments for RBL and HEPG2 cells.

Characterization of Ceramide Synthesis Inhibition by FTY720

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Finally, to determine that the changes in ceramide synthesisin cultured cells were not due to the conversion of FTY720 toFTY720-P (9), which could bind to an S1PR at the cell surfaceand thus influence ceramide synthesis, we examined the effectof FTY720 in the presence of suramin, an inhibitor of G-pro-tein-coupled receptors (31). No changes were observed in cer-amide synthesis after incubationwith FTY720 in the absence orpresence of suramin (Fig. 8A). We next incubated cells with(R)-FTY-phosphonate, a nonhydrolyzable analog of FTY-P(Fig. 1), at concentrations (1�M) that would saturate binding toan S1PR. There was no difference in the extent of inhibition ofceramide synthesis by FTY720 in the absence or presence of(R)-FTY-phosphonate (Fig. 8B). Thus, we can exclude the pos-sibility that the modulation of ceramide synthesis by FTY720 isrelated to its metabolism to FTY720-P and binding to S1PRs.

DISCUSSION

In the current study we demonstrate that FTY720 can inter-fere with SL metabolism via modulation of ceramide synthesis.

Previously, FTY720 was thought to largely mediate its biologi-cal effects after its conversion to FTY720-P and subsequentbinding to S1PRs. We now propose an additional mode ofaction, namely via modulation of ceramide synthesis.The mechanism by which FTY720 modulates ceramide syn-

thesis is surprisingly complicated. FTY720 inhibits CerS activ-ity in vitro but under certain conditions activates ceramide syn-thesis in cultured cells. The reasons for these differences are notknown, but FTY720 is not the first compound that inhibitsCerSactivity in vitro and has an opposite effect in cells. For instance,in cells overexpressingCerS, fumonisin B1, a well characterizedCerS inhibitor in vitro (32), has little or no inhibitory effect andin some cases results in elevated ceramide levels (18, 19). Theacyl chain length profile of the ceramides whose synthesis iselevated in cultured cells by fumonisin B1 is the same as the

FIGURE 4. Dependence on acyl-CoA chain length of inhibition of CerSactivity by FTY720. A, CerS activity was assayed in homogenates (100 �g ofprotein) of HEK cells using 0.25 �Ci of [4,5-3H]sphinganine/15 �M sphinga-nine, 20 �M defatted BSA and 50 �M of either C18- (for CerS1) or C16-acyl-CoA(for CerS5). B, CerS4 activity was assayed in homogenates (100 �g of protein)from CerS4-overexpressing cells using 0.25 �Ci of [4,5-3H]sphinganine, 15 �M

sphinganine, 20 �M defatted BSA, and 50 �M of either C18- or C20-acyl-CoA.The specific activity of CerS4 in the absence of FTY720 toward C18-CoA was444 pmol of ceramide formed per min per mg of protein and 389 pmol ofceramide formed per min per mg of protein toward C20-CoA in a typicalexperiment. The results are the means � S.D. of two independent experi-ments, each of which was repeated at least three times with similar results.

FIGURE 5. Kinetic analysis of CerS4 inhibition by FTY 720. Homogenates(100 �g of protein) were prepared from HEK cells overexpressing CerS4 andincubated with (squares) or without (circles) 25 �M FTY720. A, CerS4 activitywas assayed using 50 �M C18-CoA and increasing amounts of [4,5-3H]sphin-ganine/sphinganine, 20 �M defatted bovine serum albumin (such that theratio of [4,5-3H]sphinganine to sphinganine was the same at each sphinga-nine concentration). The data were plotted according to Lineweaver-Burk.The slopes of the lines were parallel, with values of 0.0169 � 0.0019 and0.0167 � 0.0019 with and without FTY720, respectively, demonstrating thatFTY inhibits CerS4 activity toward sphinganine via uncompetitive inhibition.The results are shown for a typical experiment repeated three times. B, CerS4activity was assayed using 0.25 �Ci of [4,5-3H]sphinganine, 15 �M sphinga-nine, 20 �M defatted bovine serum albumin, and increasing amounts of C18-acyl-CoA. The data were plotted according to the best fit of a sigmoid dose-response curve, which gave r2 values of 0.99 for both curves, and demonstratethat FTY720 inhibits CerS4 via noncompetitive inhibition toward C18-acyl-CoA. The inset shows a Lineweaver-Burk plot from a different experiment inwhich defatted bovine serum albumin was not included in the reactionbuffer. Noncompetitive inhibition toward C18-acyl-CoA is also observed. Theresults are the means � S.D. from two independent experiments repeatedfive or six times.

Characterization of Ceramide Synthesis Inhibition by FTY720

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profile of the ceramideswhose synthesis is inhibited in vitro (18,19), demonstrating a direct connection between the apparentlydisparate effects of fumonisin B1. In contrast to the dual effectsof fumonisin B1, which was only observed in cells overexpress-ing CerS, FTY720 elevates ceramide levels in untransfectedcells (Fig. 7). Recently, another sphingosine analog, spisulosine(33), was shown to induce de novo ceramide synthesis in pros-tate tumor cell lines (33), whereas it inhibits CerS activity invitro.4A clue concerning the differences observed between the in

vitro analysis and experiments in cultured cells might be foundin the mode of inhibition of CerS activity by FTY720. BecauseFTY720 is a sphingosine analog, we predicted that FTY720would act as a competitive inhibitor toward sphinganine, hav-ing little if any effect toward acyl-CoA. This prediction provedto be wrong, because we found that FTY720was an uncompeti-tive inhibitor toward sphinganine and a noncompetitive inhib-itor toward acyl-CoA (Fig. 5). Uncompetitive inhibitors bind toenzymes only after formation of the E-S complex (34). This type ofinhibition is most commonly encountered in multi-substratereactions where the inhibitor is competitive with respect to onesubstrate but uncompetitive with respect to another. CerS arebi-substrate enzymes, requiring the binding of both sphinga-nine and acyl-CoA for N-acylation of the former. Nothing isknown about the active site of any CerS or about the order ofbinding of the substrates in the reaction pathway. Our data

imply that sphinganine first binds toCerS to form anE-S (CerS-sphinganine) complex, and only after formation of this complexcan FTY720 bind (Fig. 9). Alternatively, there may be two sph-inganine-binding sites that act allosterically with respect to oneanother, or CerS themselves may form dimers that interactallosterically. The possibility of allosteric interactions is alsosupported by the noncompetitive mode of inhibition of acyl-CoA binding by FTY720. Noncompetitive inhibitors bind at anallosteric site on the enzyme and leave the active site unblocked(34). Thus, the binding sites of FTY720 and acyl-CoA appear tobe distinct, but the interaction between sphinganine bindingand FTY720 binding nevertheless impacts the rate of the reac-tion with respect to acyl-CoA. Finally, the experiment showingthat FTY720 inhibits ceramide synthesis at high sphinganineconcentrations in vivo, but not at low concentrations (Fig. 6),supports a complex, possibly allosteric mode of interactionbetween sphinganine and FTY720 and is consistent withuncompetitive inhibitors being most effective at high substrateconcentrations (35); this explanation might also be relevant for4 The EC50 of spisulosine toward CerS4 in vitro was �25 �M (data not shown).

FIGURE 6. Effect of FTY720 on ceramide synthesis in cultured cells. A, HEKcells overexpressing CerS1 and CerS4 or mock-transfected HEK and RBL-2H3cells were incubated with 25 �M FTY720 for 90 min. The cells were incubatedwith 1 �Ci of [4,5-3H]sphinganine, 2 �M sphinganine (for HEK cells) or with 10�M sphinganine (for RBL-2H3 cells) during the last 30 min of the incubation.The results are the means � S.D. of two independent experiments for HEKcells (repeated at least three times) and the means � S.D. for RBL cells (n � 3).B, HEK cells were incubated with 25 �M FTY720 for 90 min. The cells wereincubated with increasing amounts of [4,5-3H]sphinganine/sphinganine(such that the ratio of [4,5-3H]sphinganine to sphinganine was the same ateach sphinganine concentration) during the last 30 min of the incubationperiod. The results are the means � S.D. of three independent experiments.

FIGURE 7. SL composition of HEK cells treated with FTY720. The fatty acidcomposition of Cer, hexosylceramide, and sphingomyelin was measuredafter 90 min of incubation with 25 �M FTY720. The inset in the top panel showsthe sphinganine levels and total ceramide levels. The results are the means �S.D. (n � 3). *, p � 0.05, comparing cells treated with FTY720 versus the respec-tive untreated control. No S.D. value is given for total ceramide levels (in theinset) because this value is the sum of ceramide species with different acylchain lengths.

Characterization of Ceramide Synthesis Inhibition by FTY720

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the mode of interaction between sphinganine and other sphin-ganine analogs, such as spisulosine and fumonisin B1.The range of concentrations (low micromolar) needed to

inhibit CerS activity by FTY720 is considerably higher than thatneeded to bind and activate (mid nanomolar) S1P receptorsafter phosphorylation of FTY720 to FTY720-P. However, thiscannot exclude the possibility that some of the biological effectsof FTY720 may be mediated by FTY720 itself, prior to its me-tabolism to FTY720-P, or after its dephosphorylation by eitherlipid phosphate phosphatase 3 or lipid phosphate phosphatase1� (Fig. 1B). A number of studies have examined the biologicaleffects of FTY720 using micromolar concentrations (4, 15,36–42), and these studies are consistent with the notion thatthe effects of FTY720 that are mediated via binding to S1PRsoccur in the range expected of receptor-mediated signalingpathways, whereas additional and novel effects of FTY720, suchas the pathway described herein, which is independent of S1PRbinding, occur at much higher concentrations.Whether FTY720 ever reaches the concentrations used in

the current study (and in the study of Berdyshev et al. (42); seebelow) after its administration to either animals or to human

patients is not known. In at least one study, FTY720 was shownto accumulate at levels of �1 �M in various rat and dog tissuessoon after its injection (43). However, because FTY720 ishydrophobic, its concentrationwithin intracellularmembranesmight be much higher. The precise concentrations to whichCerS enzymesmay be exposed after FTY720 treatment is there-fore not known, and the clinical relevance of our data to under-standing the mode of FTY720 action would be strengthened bydetermination of the localmembrane concentration of FTY720in, for instance, the endoplasmic reticulum, where the CerSenzymes are located.While this manuscript was under review, a study was pub-

lished by Berdyshev et al. (42) demonstrating inhibition of CerSactivity by FTY720. In contrast to our study, Berdyshev et al.(42) reported competitive inhibition of CerS with respect tosphinganine and did not include any information on the modeof inhibition toward acyl-CoA, because the competitive modeof inhibition toward sphinganine would imply direct competi-tion between sphinganine and FTY720, alleviating the need toexamine inhibition toward acyl-CoA. In contrast, our studysuggests amuchmore complexmode of inhibition, as discussedabove. The conclusion by Berdyshev et al. (42) is based on CerSassays that do not appear to have been performed under opti-

FIGURE 8. Effect of suramin and FTY720-phosphonate on inhibition ofCerS activity by FTY720. HEK cells were incubated with suramin (50 �M, 10min) (A) or with FTY-phosphonate (FTY-P) (1 �M, 30 min) prior to incubationwith FTY720 (25 �M, 90 min). The cells were incubated with 1 �Ci of [4,5-3H]sphinganine/2 �M sphinganine during the last 30 min of the incubation.The results are the means � S.D. (n � 4) for A and the means for B (n � 2).

FIGURE 9. Possible mode of interaction of FTY720 with CerS. The datapresented in this study are consistent with the idea that sphinganine firstbinds to CerS and that subsequent binding of FTY720 leads to an allostericchange that prevents binding of acyl-CoA. See “Discussion” for further details.

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mal conditions. For example, concentrations of acyl-CoA wereonly 5 �M, which is significantly below the amount required toelicit a maximal rate of the reaction (24). Likewise, a sphinga-nine concentration of 100 nM was used, which is significantlylower than the Km of the reaction (which is in the range of 2–5�M) (26). Moreover, the effect of FTY720 reported by Berdy-shev et al. (42) might be related to the 100-fold molar excess ofinhibitor (i.e. 10 �M FTY720) versus substrate (100 nM sphinga-nine), which could lead to displacement of sphinganine fromthe BSA complex, a possibility that we excluded in our study(Fig. 2), and thus lead to apparent competitive inhibition.Clearly, conclusions concerning modes of kinetic inhibitioncannot be accurately derived from reactions performed undersuboptimal assay conditions. Thus, the data in our study sug-gest a completely different mode of action of FTY720, as illus-trated in Fig. 9.One other major difference between the two studies is our

analyses of the dependence of the inhibition by FTY720 onacyl-CoA chain length. Whereas Berdyshev et al. (42) did notobserve any significant dependence on acyl-CoA chain length,perhaps due to some of the kinetic considerations discussedabove, we observed that FTY720 inhibits ceramide synthesisusing C18-CoA to a greater extent than other acyl-CoAs. Thismay be of relevance for understanding the mode of action ofFTY720 as a modulator of ceramide synthesis in tissues thatexpress different CerS enzymes and thus contain ceramideswith different acyl chain lengths. Finally, our study examinedthe effect of FTY720 onCerS activity in three different cell lines,including RBL-2H3 mast cells, lending mechanistic signifi-cance to our studywith relation to the possible effect of FTY720in immunomodulation.In summary, we have shown a multifaceted mode of interac-

tion between FTY720 and CerS. This may be of importance forunderstanding the mode of action of FTY720, particularlybecause FTY720 is currently under investigation in a number ofclinical trials, which apparently all assume that conversion toFTY720-P is necessary for its clinical efficacy. Our currentstudy suggests a newmechanistic interpretation for some of theobserved effects of FTY720, namely viamodulation of ceramidesynthesis.

Acknowledgments—We thank Dr. Adi Mesika for help in the initialstages of this study and Dr. Al Merrill for the ESI-MS/MS analyses.

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